Thermal Cycler For PCR

ABSTRACT

An assembly for cycling vials of reaction mixtures through a series of temperature excursions is provided. The assembly can include a sample block for receiving the vials, a plurality of thermoelectric devices, a heat sink, and a clamping mechanism positioned to clamp the thermoelectric devices between the sample block and heatsink. The assembly can also include a thermal connector having a first end and a second end, the first end in close contact with the sample block and the second end in close contact with the heatsink to provide a thermal path between the sample block and the heatsink, the thermal connector being positioned to reduce thermal gradients across the sample block. The assembly can further include means for connecting the assembly to a computing apparatus for controlling the temperature excursions of the assembly.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/057,960, filed Feb. 15, 2005, which is adivisional application of U.S. patent application Ser. No. 09/075,392,filed May 8, 1998, (now U.S. Pat. No. 7,133,726 B1), which is acontinuation-in-part of PCT/US98/06189, filed Mar. 30, 1998, whichclaims benefit of U.S. Provisional Application No. 60/046,122, filed May9, 1997, and claims benefit of U.S. Provisional Application No.60/041,754, filed Mar. 28, 1997.

FIELD OF THE INVENTION

This invention pertains to the field of computer controlled instrumentsfor performing the Polymerase Chain Reaction (PCR). More particularly,the invention pertains to automated instruments that perform thereaction simultaneously on many samples and produce very precise resultsby using thermal cycling.

BACKGROUND OF THE INVENTION

The background of the invention is substantially as stated in U.S. Pat.No. 5,475,610 which is herein incorporated by reference.

To amplify DNA (Deoxyribose Nucleic Acid) using the PCR process, it isnecessary to cycle a specially constituted liquid reaction mixturethrough several different temperature incubation periods. The reactionmixture is comprised of various components including the DNA to beamplified and at least two primers sufficiently complementary to thesample DNA to be able to create extension products of the DNA beingamplified. A key to PCR is the concept of thermal cycling: alternatingsteps of melting DNA, annealing short primers to the resulting singlestrands, and extending those primers to make new copies ofdouble-stranded DNA. In thermal cycling the PCR reaction mixture isrepeatedly cycled from high temperatures of around 90.degree. C. formelting the DNA, to lower temperatures of approximately 40.degree. C. to70.degree. C. for primer annealing and extension. Generally, it isdesirable to change the sample temperature to the next temperature inthe cycle as rapidly as possible. The chemical reaction has an optimumtemperature for each of its stages. Thus, less time spent at non optimumtemperature means a better chemical result is achieved. Also a minimumtime for holding the reaction mixture at each incubation temperature isrequired after each said incubation temperature is reached. Theseminimum incubation times establish the minimum time it takes to completea cycle. Any time in transition between sample incubation temperaturesis time added to this minimum cycle time. Since the number of cycles isfairly large, this additional time unnecessarily heightens the totaltime needed to complete the amplification.

In some previous automated PCR instruments, sample tubes are insertedinto sample wells on a metal block. To perform the PCR process, thetemperature of the metal block is cycled according to prescribedtemperatures and times specified by the user in a PCR protocol file. Thecycling is controlled by a computer and associated electronics. As themetal block changes temperature, the samples in the various tubesexperience similar changes in temperature. However, in these previousinstruments differences in sample temperature are generated bynon-uniformity of temperature from place to place within the samplemetal block. Temperature gradients exist within the material of theblock, causing some samples to have different temperatures than othersat particular times in the cycle. Further, there are delays intransferring heal from the sample block to the sample, and those delaysdiffer across the sample block. These differences in temperature anddelays in heat transfer cause the yield of the PCR process to differfrom sample vial to sample vial. To perform the PCR process successfullyand efficiently, and to enable so-called quantitative PCR, these timedelays and temperature errors must be minimized to the greatest extentpossible. The problems of minimizing non-uniformity in temperature atvarious points on the sample block, and time required for and delays inheat transfer to and from the sample become particularly acute when thesize of the region containing samples becomes large as in the standard 8by 12 microtiter plate.

Another problem with current automated PCR instruments is accuratelypredicting the actual temperature of the reaction mixture duringtemperature cycling. Because the chemical reaction of the mixture has anoptimum temperature for each or its stages, achieving that actualtemperature is critical for good analytical results. Actual measurementof the temperature of the mixture in each vial is impractical because ofthe small volume of each vial and the large number of vials.

SUMMARY OF THE INVENTION

According to the invention, there is provided an apparatus forperforming the Polymerase Chain Reaction comprising an assembly capableof cycling samples through a series of temperature excursions, a heatedcover and a computer to control the process.

The invention further encompasses a sample block with low thermal massfor rapid temperature excursions. The sample block is preferablymanufactured from silver for uniform overall heat distribution and has abottom plate for uniform lateral heat distribution. In addition, tofurther offset heat losses and resulting temperature gradients from thecenter to the edges, a center pin is used as a conducting path to a heatsink.

The invention also provides a method and apparatus for achieving rapidheating and cooling using Peltier thermoelectric devices. These devicesare precisely matched to each other. They are constructed using die cutalumina on one side to minimize thermal expansion and contraction. Thedevices are constructed of bismuth telluride using specific dimensionsto achieve matched heating and cooling rates. They are designed usingminimal copper thicknesses and minimal ceramic thicknesses to furtherreduce their heat load characteristics and are assembled using aspecific high temperature solder in specified quantities.

The invention is also directed to a heatsink constructed with aperimeter trench to limit heat conduction and losses from its edges.Furthermore, the heatsink has an associated variable speed fan to assistin both maintaining a constant temperature and in cooling.

The invention is also directed to a clamping mechanism to hold thesample block to the heat sink with the thermoelectric devices positionedin between. The mechanism is designed to provide evenly distributedpressure with a minimal heat load. The design allows the use of thermalgrease as an interface between the sample block, and the thermoelectricdevices and between the thermoelectric devices and the heatsink.

There is also provided a perimeter heater to minimize the thermalnon-uniformity across the sample block. The perimeter heater ispositioned around the sample block to counter the heat loss from theedges. Power is applied to the heater in proportion to the sample blocktemperature with more power applied when the sample block is at highertemperatures and less power applied when the sample block is at lowertemperatures.

There is also provided a heated cover, designed to keep the sample tubesclosed during cycling and to heat the upper portion of the tubes toprevent condensation. The heated cover applies pressure on the sampletube cap perimeter to avoid distorting the cap's optical qualities. Thecover is self-aligning, using a skirt which mates with a sample tubetray.

The invention is also directed to a method and apparatus for determiningan ideal temperature ramp rate which is determined so as to takeadvantage of sample block temperature overshoots and undershoots inorder to minimize cycle time.

The invention also includes a method and apparatus for characterizingthe thermal power output from the thermoelectric cooling devices toachieve linear temperature control and linear and non-linear temperatureramps.

The invention is further directed to a method for predicting the actualtemperature of the reaction mixture in the sample vials at any giventime during the PCR protocol.

The invention also includes a method and apparatus for utilizingcalibration diagnostics which compensate for variations in theperformance of the thermoelectric devices so that all instrumentsperform identically. The thermal characteristics and performance of theassembly, comprised of the sample block, thermoelectric devices andheatsink, is stored in an on-board memory device, allowing the assemblyto be moved to another instrument and behave the same way.

The invention further includes a method and apparatus for measuring theAC resistance of the thermoelectric devices to provide early indicationsof device failures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional view of a portion of the sample blockaccording to the invention.

FIG. 2 is an enlarged, isometric view of a thermoelectric deviceconstructed according to the invention.

FIG. 2A is a side, elevational view of a thermoelectric deviceconstructed according to the invention.

FIG. 3 is a cut-away, partial, isometric view of the heatsink accordingto the invention.

FIG. 4 is an exploded view of an assembly including a sample block,thermoelectric devices and heatsink.

FIG. 5 is an isometric view of the heated cover in accordance with theinvention.

FIG. 6 is a chart depicting the Up Ramp (heating rate) vs. Power.

FIG. 7 is a chart depicting the Down Ramp (cooling rate) vs. Power.

FIG. 8 is a chart for predicting and compensating for temperatureovershoots and undershoots in accordance with the invention.

FIG. 9 is a block diagram of the AC resistance measurement circuit ofthe invention.

FIG. 10 shows a perimeter heater and its location surrounding the sampleblock.

FIG. 11 is a detailed view of the perimeter heater of FIG. 10.

FIG. 12 shows the power applied to the perimeter heater as a function ofthe temperature of the sample block.

FIG. 13 shows a thermal model of a sample in a sample vial.

FIG. 14 is an illustration of the initial conditions of the thermalmodel of FIG. 13.

FIG. 15 shows the sample block and a seal designed to protect thethermoelectric devices from the environment.

DETAILED DESCRIPTION OF THE INVENTION

Generally, in the case of PCR, it is desirable to change the sampletemperature between the required temperatures in the cycle as quickly aspossible for several reasons. First the chemical reaction has an optimumtemperature for each of its stages and as such less time spent atnon-optimum temperatures means a better chemical result is achieved.Secondly a minimum time is usually required at any given set point whichsets a minimum cycle time for each protocol and any time spent intransition between set points adds to this minimum time. Since thenumber of cycles is usually quite large, this transition time cansignificantly add to the total time needed to complete theamplification.

The absolute temperature that each reaction tube attains during eachstep of the protocol is critical to the yield of product. As theproducts are frequently subjected to quantitation, the product yieldfrom tube to tube must be as uniform as possible and therefore both thesteady-state and dynamic thermal uniformity must be excellent across theblock.

Heat-pumping into and out of the samples is accomplished by usingPeltier thermoelectric devices. These are constructed of pellets ofn-type and p-type bismuth telluride connected alternately in series. Theinterconnections between the pellets is made with copper which is bondedto a substrate, usually a ceramic (typically alumina).

The amount of heat-pumping required is dependent on the thermal load andthe ramp rate, that is, the rate at which the temperature is required tochange. The sample tube geometry and sample volumes are not variables asthe sample tubes are established as an industry standard, fitting intomany other types of instruments such as centrifuges. The sample volumeis defined by user need. Therefore the design variables primarily affectthe sample block, thermoelectric devices, heatsink, fan and the thermalinterface media between the thermoelectric devices and both the heatsinkand the sample block.

The block geometry must also meet the necessary thermal uniformityrequirements because it is the primary contributor to lateral conductionand therefore evens out any variation in thermal uniformity of thethermoelectric coolers themselves. The conflicting requirements of rapidramp rates (indicating low thermal mass) and high lateral conduction(indicating a large material mass) are met by concentrating the bulk ofthe block structure in a base plate, and minimizing the thermal mass ofthe upper portion of the block which holds the sample tubes. The optimalmaterial for block fabrication is pure silver which has relatively lowthermal mass and very good thermal conduction. Silver also lends itselfwell to electroforming. In practice the optimal block geometry has alight electroformed upper portion to hold the sample tubes fixed to arelatively thick base plate which provides lateral conduction. Thethermal mass of the block is concentrated in the base plate where thematerial contributes the most to thermal uniformity. The electroformedportion of the block has a minimum thickness which is defined by twoparameters: first, the material cannot be so thin as to make it toodelicate for normal handling; second, the wall thickness is required toconduct heat out of the upper regions of the sample tube. Circulation inthe sample itself is achieved by convection inside the tube and sampletemperature is relatively uniform along the height of the tube, but goodthermal conductivity between the tube walls and the base plate increasesthe effective surface area available for conduction of heat between thesample and the base plate. The base plate thickness has a minimum valuedefined by lateral conduction requirements which is a function of thethermal uniformity of the thermoelectric coolers and structuralrigidity.

Another contributor to the thermal mass is the alumina ceramic layerswhich form part of the structure of the thermoelectric cooler itselfThere are two alumina layers in the construction of the thermoelectriccooler, one on the sample block side and another on the heatsink side.The thickness of the layers should be minimized as much as possible, inthis case the practical limit of thinness for the alumina thickness isdefined by the manufacturing requirements of thermoelectric coolerfabrication. This particular layer of ceramic could in principal bereplaced by a different layer altogether such as a thin sheet of Kaptonwhich would reduce the thermal mass even more, but at the present timealthough coolers are available with this structure, reliability isunproven. It is anticipated that once the technology has been developedfurther, then a cooler of such a design may be preferred. However, thethin alumina layers also contribute to system reliability.

The copper conductors within the cooler are a significant thermal loadand are not overlooked in the design of the system. The thickness of thecopper traces is defined by the requirement of carrying current throughthe device. Once the current is known the required copper thickness canbe calculated.

Sample Block

FIG. 1 shows a cross sectional view of a portion of the sample block 36which typically has 96 wells 20, each for receiving a sample vial. Thesample block is constructed of silver and comprises an upper supportplate 21 and the sample wells 20 electroformed as one piece fastened toa base plate 22. The base plate 22 provides lateral conduction tocompensate for any difference in the thermal power output across thesurface of each individual thermoelectric device and for differencesfrom one thermoelectric device to another.

There are always boundary losses in any thermal system. In a rectangularconfiguration there is more heat loss in the corners. One solution is touse a round sample block, but the microtiter tray format that is incommon usage is rectangular and this must be used to retaincompatibility with other existing equipment. Once the edge effects havebeen eliminated using all standard means, such as insulation etc., thereremains a tendency for the center of the sample block to be warmer thanthe corners. Typically it is this temperature difference that definesthe thermal uniformity of the sample block. In accordance with theinvention, the center temperature is reduced by providing a smallthermal connection from the center of the sample block to the heat sink.By using a pin 24 which acts as a “heat leak” in the center of thesample block, the temperature gradient across the sample block can bereduced to an acceptable level. The amount of conduction required isquite small and a 1.5 mm diameter stainless steel pin has been found tobe sufficient. Moreover, a pin made of the polymer ULTEM, manufacturedby General Electric may also be used. As more fully described below, thepin also serves to help position and lock into place components of theassembly illustrated in FIG. 4.

Peltier Thermoelectric Devices (TEDs)

Thermal uniformity of the sample block is critical to PCR performance.One of the most significant factors affecting the uniformity isvariations in the thermoelectric device performance between devices. Themost difficult point at which to achieve good uniformity is during aconstant temperature cycle far from ambient. In practice this is aconstant temperature cycle at approximately 95.degree. C. Thethermoelectric devices are matched tinder these conditions to make a setof devices for each heatsink assembly which individually produce thesame temperature for a given input current. The thermoelectric devicesare matched to within 0.2.degree. C. in any given set, this value beingderived from the maximum discrepancy that can be rectified by thelateral conduction of the sample block baseplate.

FIG. 2A shows a side view of a typical Peltier thermal electric device60. The device is composed of bismuth telluride pellets 30, sandwichedbetween two alumna layers 26. The pellets are electrically connected bysolder joints 28 to copper traces 29 plated onto the alumina layers. Onealumina layer has an extension 31 to facilitate electrical connections.The thickness of the extended areas is reduced to decrease the thermalload of the device.

FIG. 2 shows an isometric view of a typical Peltier thermoelectricdevice. The alumina layer 26 that forms the outer wall of thethermoelectric device, expands and contracts during temperature cyclingat a different rate than the sample block 19. The motion of the aluminais transmitted directly to the solder 28 connecting the internal bismuthtelluride pellets 30. This motion can be reduced dramatically by cuttingthe alumina into small pieces 32 called die so that the field ofexpansion is small. The minimum size of the die is defined by the sizeof the copper traces required to carry current through thethermoelectric device and the requirements that the device retain somestrength for handling.

Using thin alumina layers in the thermal electric device (of the orderof 0.508 mm) not only reduces the thermal load but also means that for agiven required heat pumping rate the temperature that the ends of thepellet reaches is reduced due to the increase in thermal conductivity k.This enhances reliability by reducing the thermal stress on the solderjoint.

Generally in PCR the reaction temperatures are above ambient and in therange 35 to 96.degree. C. In the most important cases the block isheated or cooled between two above ambient temperatures where the flowof heat due to conduction is from the block to the heat sink. The key tooptimizing the system cycle time, given an optimized blockconfiguration, is to balance the boost to the ramp rate when coolingprovided by the conduction, against the boost provided to the heatingramp rate by the Joule effect of resistance heating.

If the cross-section of the bismuth telluride pellets in a giventhermoelectric device were considered constant, the heating ramp ratewould be increased by increasing the height of the pellet. This isbecause the conduction path through the thermoelectric device would bemade longer thereby decreasing k. This also has the effect of reducingthe current required to maintain a given block temperature in the steadystate. During the down ramp, i.e. cooling the block, the decreased kmeans that the conduction contribution will be reduced and so the downramp rate will be reduced.

Conversely, if the height of the bismuth telluride pellet were to bedecreased for a given cross-section, then k would be increased. Thiswould increase the current required to maintain an elevated temperaturein the steady state and would increase the cooling ramp rate. Heatingramp rates would be reduced as a larger portion of the heat in the blockwould be conducted directly to the heat sink. Decreasing the bismuthtelluride pellet height also increases the holding power required for agiven temperature due to the losses through the thermoelectric devicesand reduces the thermal load, increasing the maximum possible ramp ratefor given power. Therefore the optimized thermoelectric device can bederived by adjusting the height of the Bismuth Telluride pellets untilthe heating rate matches the cooling rate.

The ratio 1:A for the pellets also defines the resistance of the devicei.e. R=nr(h/A) where n is the number of pellets, r is the resistivity ofthe Bismuth Telluride being used, h is the height of the pellet and A isthe cross-sectional area.

The resistance must be measured as an AC resistance because of theSeebeck effect. Because the geometry defines the resistance of thedevice, another design boundary is encountered in that the device mustuse a cost effective current to voltage ratio because too high a currentrequirement pushes up the cost of the amplifier. The balanced solutionfor the silver electroformed block described above is: [0053] Pelletheight=1.27 mm [0054] Pellet cross-sectional area=5.95 mm.sup.2

If the thermal cycler was to be used as part of another instrument, e.g.integrated with detection technology, then it may be more convenient touse a different current source which would lead to a modifiedthermoelectric device geometry. The current source in the presentembodiment consists of a class D type switch-mode power amplifier with acurrent sensing resistor in series with the device and ground.

Because the thermoelectric devices are soldered together, excess soldercan wick up the side of the bismuth telluride pellets. Where thisoccurs, k is increased which results in a local cold spot, also called amild spot. These cold spots are reduced in number and severity byapplication of the minimum amount of solder during the assembly processof the thermal electric device. For the same reason, it is alsonecessary to ensure that the solder used to attach the connecting wiresto the thermoelectric device does not contact the pellet

High temperature solder has been shown to not only have improved hightemperature performance but it is also generally more resistant tofailure by stress reversals and hence is most appropriate in thisapplication. The solder used in this invention may be of the type asdescribed in U.S. Pat. No. 5,441,576.

Heatsink

FIG. 3 shows the heatsink 34 assembled with the thermoelectric devices39 and the sample block 36. A locating frame 41 is positioned around thethermoelectric devices to align them with the sample block and theheatsink to ensure temperature uniformity across the sample block. Theframe is composed of Ultem or other suitable material and has tabs 43 atits corners to facilitate handling. The heatsink 34 has a generallyplaner base 34 and fins 37 extending from base 35. The thermal mass ofthe heat sink is considerably larger than the thermal mass of the sampleblock and samples combined. The sample block and samples together have athermal mass of approximately 100 joules/.degree. K and that of the heatsink is approximately 900 joules/.degree. K. This means that the sampleblock clearly changes temperature much faster than the heat sink for agiven amount of heat pumped. In addition the heat sink temperature iscontrolled with a variable speed fan as shown in FIG. 9. The temperatureof the heat sink is measured by a thermistor 38 placed in a recess 40within the heatsink and the fan speed is varied to hold the heat sink atapproximately 45.degree. C. which is well within the normal PCR cyclingtemperature range, where maintaining a stable heat sink temperatureimproves the repeatability of system performance. When the blocktemperature is set to a value below ambient then the heat sink is set tothe coolest achievable temperature to reduce system power consumptionand optimize block thermal uniformity. This is accomplished simplyoperating the fan at full speed.

The heat sink temperature measurement is also used by the thermoelectricdevice control algorithm described below in linearizing the thermaloutput power from the thermoelectric devices.

The heatsink temperature uniformity is reflected in the uniformity ofthe block temperature. Typically the heatsink is warmer in the middlethan it is at the edges and this adds to other effects that lead to thecorners of the block being the coldest. A trench 44 is cut into the heatsink outside the perimeter of the thermoelectric device area to limitthe conduction of heat and decreases edge losses from the area boundedby the trench.

Thermal Interface and Clamping Mechanism

Thermoelectric device manufacturers recommend that thermoelectricdevices be held under pressure to improve life-expectancy. (The pressurerecommended is often defined by the thermal interface media selected.)The pressure that is recommended varies from manufacturer tomanufacturer but is in the range of 30 to 100 psi for cyclingapplications.

There are many thermal interface media available in sheet form which canbe used to act as a compliant layer on each side of the thermoelectricdevices, but it has been demonstrated that thermal grease gives farsuperior thermal performance for this application. Unlike othercompliant sheets which have been shown to require 30 psi or more evenunder optimal conditions, thermal grease does not require high pressureto ensure that good thermal contact has been made. Also thermal greaseacts as an effective lubricant between the expanding and contractingsilver block and the thermoelectric device surface, enhancinglife-expectancy. Thermalcote II thermal grease manufactured byThermalloy, Inc. may be used.

Because the silver block is relatively flexible and son it cannottransmit lateral clamping pressure very effectively. However, becausethe thermal interface media is thermal grease, the clamping forcerequired is low.

FIG. 4 shows an exploded view of the assembly with the preferredembodiment of the clamping mechanism. Each clamp 46 is made up of aseries of fingers 48 extending from a spine 49. The fingers 48 aresized, shaped and spaced so as to fit between the wells 20 of the sampleblock 36 and thus apply pressure at a corresponding series of points onthe base plate 22 of the sample block 36. The open honeycomb structureof the electroformed sample wells allows the fingers to be inserted somedistance into the block, thereby applying the pressure more evenly thanan edge clamping scheme would. These fingers apply pressure at a seriesof local points to minimize the contact area between the mass of theclamp and the sample block so that the clamp does not add significantlyto the thermal load. The clamps are molded from a glass filled plasticwhich has the necessary rigidity for this application. The pressure isapplied by deforming the fingers with respect to mounting posts 50 whichmay be separate clamp structures, but are preferably integrally formedwith the clamps 46. The clamps 46 are held flush to the surface of theheat sink with a series of screws 52 extending through correspondinghole 53 in clamps 46 and then into threaded holes 55 in heatsink 34.This scheme eliminates the necessity to set the pressure with adjustmentscrews as the clamps can simply be tightened down by standard torquingtechniques.

The resulting even pressure distribution ensures that the full area ofthe thermoelectric devices is in good thermal contact with the block andthe heatsink reducing local thermal stresses on the thermoelectricdevices.

FIG. 4 shows other important features of the invention. A printedcircuit board 82 includes a memory device 96 for storing data andsurrounds the thermoelectric devices and provides electricalconnections. Alignment pins 84 are seated in holes 86 in the heatsinkand protrude through alignment holes 88 to align the printed circuitboard with the heatsink. The locating frame 41 is positioned around thethermoelectric devices and has a cross beam 90 with a through hole 92.Pin 24 (shown in FIG. 1) fits into a hole (not shown) in the sampleblock, extends through hole 92 in the locating frame and further extendsinto hole 94 in the heatsink.

Perimeter Heater

In order to bring the temperature uniformity across the sample block toapproximately .+−.0.2.degree. C., a perimeter heater is positionedaround the sample block to eliminate heat losses from its edges.Preferably, the heater is a film type, having low mass with insidedimensions slightly larger than the sample block. FIG. 10 shows theperimeter heater 74 and its approximate location surrounding the sampleblock 36. The heater is not fastened in place, it is simply positionedin the air around the perimeter of the sample block in order to warm theair in the immediate vicinity.

FIG. 11 shows a detailed view of the perimeter heater 74. The heater isrectangular as determined by the dimensions of the sample block and ismanufactured so that it has separate power densities in specific areasto reflect the varying amounts of heat loss around the perimeter of theblock. Matching lower power density regions 76 (0.73 W/in.sup.2) arelocated in the center portions of the short sides of the rectangle andmatching higher power density regions 78 (1.3 W/in.sup.2) are located inthe longer sides, extending into the shorter sides.

As shown in FIG. 12, the power applied to the perimeter heater isregulated to correspond to the temperature of the sample block, withmore power applied to the heater at higher block temperatures and lessapplied at lower block temperatures.

Heated Cover:

FIG. 5 shows the heated cover 57. The heated cover applies pressure tothe sample vial caps to ensure that they remain tightly closed when thesample is heated.

Further, pressure transferred to the vials assures good thermal contactwith the sample block. The cover is heated under computer control to atemperature above that of the sample to ensure that the liquid does notcondense onto the tube cap and instead remains in the bottom of the tubewhere thermal cycling occurs. This is described in U.S. Pat. No.5,475,610, mentioned above. The heated platen 54 in the presentinvention does not press on the dome of the cap but instead presses onthe cap perimeter. The platen has a surface shaped in this manner sothat optical caps are not distorted by the application of pressure.Thus, tubes that have been cycled can be directly transferred to anoptical reader without the need to change the cap.

Because the heated platen has recesses 56 in it to clear the cap domes,there is a need to align the plate to the tube positions before applyingpressure to avoid damage to the tubes. This is accomplished by use of a“skirt” 58 around the perimeter of the platen which aligns to themicrotiter tray before the plate touches the tube caps. The cover has asliding mechanism similar to that used on the PYRIS DifferentialScanning calorimeter by the Perkin Elmer Corporation allowing the coverto slide back to allow sample vials to be inserted into the sample blockand forward to cover the sample block and move down engage the vials.

Determining the Ideal Ramp Rate:

The optimized ramp rate has been empirically determined to be 4.degree.C./sec. Any system which has a higher block ramp rate than this cannotfully utilize the benefits of temperature overshoots and consequentlyachieves an insignificant reduction in cycle time.

FIG. 6 is a chart depicting the Up Ramp (heating rate) vs. Power andFIG. 7 is a chart depicting the Down Ramp (cooling rate) vs. Power.

When heating the block to a temperature above ambient, the Joule heatingand the Seebeck heat pumping both act to heat the sample block againstconduction. When cooling the block between two temperatures aboveambient, the Seebeck heat pumping and conduction act against the Jouleheating. During cooling, significant power is required to hold the blocktemperature steady against the flow of heat out or the block byconduction. Therefore even with zero power applied, the block will coolat a significant rate. As the current is increased, the Seebeck effectincreases the cooling obtained. However as the current is increasedfurther the joule effect, which is proportional to the square of thecurrent, quickly starts to take over acting against the Seebeck cooling.Therefore a point is reached where applying additional power actsagainst the required effect of cooling. In the heating mode these twoeffects act together against conduction and no ceiling is reached. Inpractice the heating power vs. input current is approximately linear.This is why the design criteria centers around meeting the cooling raterequirements; the heating rate can always be achieved by the applicationof more power.

Characterizing the Output of the TED's

The following equation describes the total heat flow from the cold sideof a thermal electric cooler.0=½*R(t.sub.avg)*I.sup.2+t.sub.c*S(t.sub.avg)*I−(k(t.sub.avg)*(t.sub.c−−t.sub.h)+Q.sub.c)where [0077] t.sub.c=cold side temperature of cooler [0078] t.sub.h=hotside temperature of cooler [0079] t.sub.avg=average of t.sub.c andt.sub.h [0080] R(t)=electrical resistance of cooler as a function oftemperature [0081] S(t)=Seebeck coefficient of the cooler as a functionof temperature [0082] K(t)=Conductance of cooler as function oftemperature [0083] I=electrical current applied to cooler [0084]Q.sub.c=total heat flow from the cold side of the cooler

Given a desired heat flow, Q.sub.c, and the hot and cold sidetemperatures, t.sub.c and t.sub.h, the equation is solved for I, thecurrent required to produce Q.sub.c. The solution of this equation isused for three purposes:

1) To achieve linear temperature transitions or ramps. For lineartemperature transitions, constant thermal power is required. To maintainconstant thermal power when temperatures t.sub.c and t.sub.h arechanging, it is necessary to solve for I in equation 1 periodically. Theresult is the current then applied to the coolers. To compensate forerrors a proportional integral derivative (PID) control loop is appliedwhere: Error input to PID=Set point Rate-Actual Rate and Output from thePID is interpreted as percent Q

2) To achieve a linear PID temperature set point control algorithm overthe desired temperature range: Input to the PID control is the errorsignal t.sub.c−Set point. Output from the PID control is interpreted asa % of Q.sub.max. Equation 1 is used to determine the current value, I,which will result in the % of Q.sub.max output by the PID control, underthe current temperature conditions.

3) To achieve non-linear temperature transitions or ramps wheretemperature transitions are defined by the derivative of temperaturewith respect to time, dT/dt, as a function of block temperature.

This function is approximated by a table containing Block temperature T,dT/dt data points in 5 C increments for cooling and by a linear equationfor heating. The small effect of sample mass on dT/dt profiles, althoughmeasurable, is ignored. Knowing the total thermal mass, MC.sub.p(joules/.degree. K), involved during temperature transitions, the amountof thermal power, Q (joules/sec), required to achieve the desired rateprofile, dT/dt (.degree. K/sec), is given at any temperature by thefollowing equation:

Q=MC.sub.p*dT/dt

The solution to equation 1 is used to determine the current value, I,which will result in the desired Q under the current temperatureconditions. This process is repeated periodically during temperaturetransitions.

Controlling Overshoot and Undershoot

There is a practical limit to the ramp rates and the resulting cycletimes that can be achieved. The sample has a time constant with respectto the block temperature that is a function of the sample tube and tubegeometry which, because the tube is an industry standard, cannot bereduced. This means that even if the sample tube wall temperature ischanged as a step function e.g. by immersion in a water bath, the samplewill have a finite ramp time as the sample temperature exponentiallyapproaches the set point. This can be compensated for by dynamicallycausing the block to overshoot the programmed temperature in acontrolled manner. This means that the block temperature is drivenbeyond the set point and back again as a means of minimizing the timetaken for the sample to reach the set point. As the possible ramp-ratesincrease, the overshoot required to minimize the time for the sample toreach the set point gets larger and a practical limit is soon reached.This occurs because although the average sample temperature does notovershoot the set point, the boundary liquid layer in the tube doesovershoot to some extent. When cooling to the priming temperature, toogreat an overshoot can result in non-specific priming. Therefore thebest advantage is to be gained in a system which utilizes this maximumramp rate combined with optimized overshoots that are symmetrical onboth up and down ramps.

FIG. 8 is a chart for predicting and compensating for temperatureovershoots and undershoots. In order to drive the block temperaturebeyond the set point and back again in a controlled fashion the systemfirst measures the block temperature, Tbn+1 and then solves thefollowing equations: Ts.sub.n+1=Ts.sub.n+(Tb.sub.n+1−Ts.sub.n)*0.174/RCTsf.sub.n=(Tb.sub.n−Ts.sub.n−mRC)(1−e.sup.−tm/RC)+mtr.sub.n+Ts.sub.nwhere Tb is the measured block temperature, Ts is the calculated sampletemperature, Tsf is the final calculated sample temperature if the blockis ramped down at time t.sub.n, R is the thermal resistance between thesample block and the sample, C is the thermal capacitance of the sample,m is the slope of a line defined by the points Tb and Tsf and tr is thetime for the sample block to return to the set point if the systemcaused it to ramp toward the set point at the same rate it is wasramping away.

If the resulting Tsf.sub.n is within a particular error window aroundthe set point then the system causes the sample block to ramp back tothe set point at the same rate it was ramping away. If the resultingTsf.sub.n is outside the particular error window then the system causesthe sample block to continue to ramp away from the set point at the samerate. While ramping back toward the set point the same proportionalintegral derivative (PID) control loop described above is applied.

Determining Sample Temperature

The temperature of a sample in a sample vial is determined by using themodel illustrated in FIG. 13 where: TB1 k is the measured baseplatetemperature; TSmp is the calculated sample temperature; TPlastic is thecalculated plastic temperature; TCvr is the measured cover temperature;R1 is the thermal resistance of the plastic vial between the block andsample mixture; C1 is the thermal capacitance of the sample mixture; R2and R3 represent the thermal resistance of air in parallel with theplastic vial between the sample mixture and the cover; and C2 is thethermal capacitance of the plastic vial between the sample mixture andthe cover.

The model above is solved for TSmp(t) and TPlastic(t) given that: TB1k=mt+TB1 k 0, TCvr=K and initial conditions are non-zero. Taking initialconditions and the slope of TB1 k to be the only variables, asillustrated in FIG. 14, the equations are refactored giving equationsfor Tsmp and TPlastic.

Given the following relationships: g1=1/R1; g2=1/R2; g3=1/R3;a=(g1+g2)/C1; b=g2/C1; f=g2/C2; g=(g2+g3)/C2;alpha=−(−g/2−a/2−(sqrt(g*g−2*g*a+a*a+4*f*b))/2); andbeta=−(−g/2−a/2+(sqrt(g*g−2*g*a+a*a+4*f*b))/2), the coefficients for thesample temperature equation become:coef1=(g3/C2)*(−b/(beta*(alpha−beta))*exp(−beta*T)+b/(alpha*beta)+(b/(alp−ha*(alpha−beta)))*exp(−alpha*T))coef2=(b/(alpha−beta))*exp(−beta*T)−(b/(alpha−beta))*exp(−alpha*T)coef3=(g1/C1)*(g/(alpha*beta)+(−alpha+g)*exp(−alpha*T)/(alpha*(alpha−beta−))+(beta−g)*exp(−beta*T)/(beta*(alpha−beta)))coef4=(g1/C1)*((g−beta)*exp(−beta*T)/(pow(beta,2)*(alpha−beta))−g/(beta*p−ow(alpha,2))+(1+T*g)/(alpha*beta)+(−g+alpha)*exp(−alpha*T)/(pow(alpha,2)*(−alpha−beta))−g/(alpha*pow(beta,2)))coef5=(−g+alpha)*exp(−alpha*T)/(alpha−beta)+(g−beta)*exp(−beta*T)/(alpha−−beta)and the coefficients for the plastic vial temperature equation become:coef6=(g3/C2)*((beta−a)*exp(−beta*T)/(beta*(alpha−beta))+a/(alpha−*beta)+(−alpha+a)*exp(−alpha*T)/(alpha*(alpha−beta)))coef7=(−beta+a)*exp(−beta*T)/(alpha−beta)+(alpha−a)*exp(−alpha*T)/(alpha−−beta)coef8=(g1/C1)*(f*exp(−beta*T)/(pow(beta,2)*(alpha−beta))−f/(beta*pow−(alpha, 2))−f*exp(−alpha*T/(pow(alpha,2)*(alpha−beta))+T*f/(alpha*beta)−f/(−alpha*pow(beta,2)))coef9=(g1/C1)*(−f*exp(−beta*T)/(beta*(alpha−beta))+V(alpha*beta)+f*exp(−−alpha*T)/(alpha*(alpha−beta)))coef10=f*exp(−beta*T)/(alpha−beta)−f*exp(−alpha*T)/(alpha−beta)and slope=(TB1 k−TB1 k 0)/T where T is the sampling period (0.174 sec)Utilizing the model in FIG. 13 then,Smp=coef1*TCvr0+coef2*TPlastic0+coef3*TB1 k 0+coef4*slope+coef5*TSmp0TPlastic=coef6*TCvr0+coef7*TPlastic0+coef8*slope+coef9*TB1 k0+coef10*TSmp0

The coefficients are recalculated at the beginning of each PCR protocolto account for the currently selected sample volume. TSmp and TPlasticare recalculated for every iteration of the control task.

To determine the sample block set point, TB1 k SP, during a constanttemperature cycle, Tb1 k is determined using the equation for TSmp. Tb1k 0=(Tsmp−coef1*TCvr0−coef2*TPlastic0−coef4*slope−coef5*TSmp0)/coef3

When maintaining a constant temperature the slope=0 andTsmp=Tsmp0=TSmpSP (sample temperature set point) and: TB1kSP=(TSmpSP−coef1*TCvr−coef2*TPlastic−coef5*TSmpSP)/coef3

The equation for TB1 kSP is solved on every pass of the control loop toupdate the sample block set point to account for changes in temperatureof the plastic and cover.

Calibration Diagnostics:

The control software includes calibration diagnostics which permitvariation in the performance of thermoelectric coolers from instrumentto instrument to be compensated for so that all instruments performidentically. The sample block, thermoelectric devices and heatsink areassembled together and clamped using the clamping mechanism describedabove. The assembly is then ramped through a series of known temperatureprofiles during which its actual performance is compared to thespecified performance. Adjustments are made to the power supplied to thethermoelectric devices and the process is repeated until actualperformance matches the specification. The thermal characteristicsobtained during this characterization process are then stored in amemory device residing on the assembly. This allows the block assemblyto be moved from instrument to instrument and still perform withinspecifications.

AC Resistance Measurement:

The typical failure mode for the thermoelectric devices is an increasein resistance caused by a fatigue failure in a solder joint. Thisresults in an increase in the temperature of that joint which stressesthe joint further, rapidly leading to catastrophic failure. It has beendetermined empirically that devices that exhibit an increase in ACresistance of approximately 5% after about 20,000 to 50,000 temperaturecycles will shortly fail. The AC resistance of the thermoelectricdevices are monitored by the instrument to detect imminent failuresbefore the device in question causes a thermal uniformity problem.

This embodiment automates the actual measurement using a feedbackcontrol system and eliminates the need to remove the thermoelectricdevice from the unit. The control system compensates for the temperaturedifference between the two surfaces of the thermoelectric device causedby the heat sink attached to one side and the sample block attached tothe other. The control system causes the thermoelectric device toequalize its two surface temperatures and then the AC resistancemeasurement is made. The micro-controller performs a polynomialcalculation at the referenced time of the AC measurement to compensatefor ambient temperature error.

FIG. 9 shows the sample block 36, a layer of thermoelectric device 60and heatsink 34 interfaced with the system microcontroller 62 andbipolar power amplifier 64. The temperature sensor is already present inthe heatsink 38 and an additional temperature sensor attached to thesample block 36 with a clip (not shown) formed of music wire areutilized to determine the temperature differential of the surfaces ofthe thermoelectric device.

The bipolar power amplifier supplies current in two directions to thedevice. Current in one direction heats the sample block and current inthe other direction cools the sample block. The bipolar power amplifieralso has signal conditioning capability to measure the AC voltage and ACcurrent supplied to the thermoelectric device. A band pass filter 68 isincorporated into the signal conditioning to separate an AC measurementsignal from the steady state signal that produces a null condition forthe temperature difference across the thermoelectric device.

The micro-controller incorporates the necessary capability to processthe measurement information and perform the feedback in real time. Italso stores the time history of the AC resistance and the number oftemperature cycles of the thermoelectric device and displays theinformation to the operator on the display 70. The AC measurement isnormally done during initial turn on. However, it can be activated whenself diagnostics are invoked by the operator using the keypad 72. Ananalog to digital and digital to analog converter along with signalconditioning for the temperature sensors and AC resistance measurementis also integrated into the micro-controller in order for it to performits digital signal processing.

Sealing the Thermoelectric Device Area from the Environment.

The thermoelectric devices are protected from moisture in theenvironment by seals and the chamber is kept dry with the use of adrying agent such is silica gel. The seal connects from the silverelectroform to the surrounding support and as such adds to the edgelosses from the block. These losses are minimized by the use of a lowthermal conductivity pressure seal 98 and by the use of the perimeterheater described above. The seal 98 has a cross-section generally in theshape of a parallelogram with several tabs 100 spaced about the lowersurface of seal 98 for holding seal 98 to the edge of the sample blockas shown in FIG. 15.

The seal 98 is installed by first applying RTV rubber (not shown) aroundthe perimeter 110 of the upper portion of the sample block. The seal 98is then placed on the RTV rubber. More RTV rubber is applied to theperimeter 120 of the seal and then a cover (not shown) is installedwhich contacts the RTV rubber-seal combination. The cover has a skirtwhich also contacts a gasket (not shown) on the printed circuit board toeffect a more effective seal.

1-18. (canceled)
 19. An apparatus for performing a polymerase chainreaction, said apparatus comprising: an assembly for cycling a pluralityof vials through a series of temperature excursions; a cover forapplying a seating force to said vials and for applying a temperature toan upper portion of said vials; and a computing apparatus forcontrolling said temperature excursions of said assembly and saidtemperature of said cover; said assembly including: a sample block forreceiving said vials; a plurality of thermoelectric devices controlledby said computing apparatus; a heat sink; a clamping mechanismpositioned so as to clamp said thermoelectric devices between saidsample block and heatsink; and a thermal connector having a first endand a second end, said first end in close contact with said sample blockand said second end in close contact with said heatsink so as to providea thermal path between said sample block and said heatsink, said thermalconnector being positioned to reduce thermal gradients across saidsample block.
 20. The apparatus of claim 19 wherein said sample blockcomprises: a plurality of sample wells for receiving sample vials, eachwell having a top and bottom; an upper support plate connecting the topsof said sample wells; and a bottom plate connecting the bottoms of saidsample wells.
 21. The apparatus of claim 20 wherein said upper supportplate and said sample wells are electroformed as a single piece.
 22. Theapparatus of claim 20 wherein said sample block is comprised of silver.23. The apparatus of claim 19 further comprising an associated memorydevice capable of storing data related to said assembly.
 24. Theapparatus of claim 19 wherein said cover comprises: a platen, verticallyand horizontally displaceable in relationship to said vials, said platenincluding: a skirt extending downward around the perimeter of saidplaten, said skirt having dimensions corresponding to the perimeter of astandard microtiter tray, said skirt constructed to engage saidperimeter of said tray during vertical displacement of said platen,applying a seating force on said vials for maintaining a snug fitbetween walls of said sample vials and said assembly for receiving saidsample vials; means for forcibly lowering said platen to maintain saidseating force; and heating means positioned in close contact with saidplaten to maintain said platen at a constant temperature.
 25. Theapparatus of claim 19, wherein the thermal connector is a pin.
 26. Theapparatus of claim 25, wherein the pin is a steel pin or polymer pin.27. The apparatus of claim 25, wherein said pin is positioned at thecenter of the sample block.
 28. The apparatus of claim 19, wherein thecover is configured for applying a constant temperature to an upperportion of said vials.
 29. An assembly for cycling vials of reactionmixtures through a series of temperature excursions, comprising: asample block for receiving vials of reaction mixtures; a plurality ofthermoelectric devices; a heat sink; a clamping mechanism positioned soas to clamp said thermoelectric devices between said sample block andsaid heatsink; a thermal connector having a first end and a second end,said first end in close contact with said sample block and said secondend in close contact with said heatsink so as to provide a thermal pathbetween said sample block and said heatsink, said thermal connectorbeing positioned to reduce thermal gradients across said sample block;and means for connecting the assembly to a computing apparatus forcontrolling said temperature excursions of said assembly.
 30. Theapparatus of claim 29, wherein the thermal connector is a pin.
 31. Theapparatus of claim 30, wherein the pin is a steel pin or polymer pin.32. The apparatus of claim 30, wherein said pin is positioned at thecenter of the sample block.